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Radicle emergence test in relation to
emergence, seed storage and other
vigour tests
Stan Matthews
University of Aberdeen
United Kingdom
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• The germination progress curve
• Mean germination time (MGT)
• MGT in relation to:
– seed age
– seed performance (vigour)
– radicle emergence (RE)
• RE and seed performance (vigour)
• RE as a vigour test
• RE in relation to other vigour tests
• Explanation of relationships
• Concluding comments
Outline
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0
20
40
60
80
100
0 24 48 72 96 120 144
Time from set to germinate (h)
Germ
ination (
%)
F
H
I
Lag period
Longer lag period
Germination progress curves to radicle emergence (RE)
Mean germination time (MGT) = Ʃnt / Ʃn
n = number of newly germinated seeds at time t
t = days from when seeds were set to germinate.
MGT= Mean length of lag period
Mean length of lag period differs between commercial seed lots
Why do the lag periods differ between lots?
Maize
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y = -0.40x + 72.01
R² = 0.850***
30
35
40
45
60 70 80 90 100
MG
T a
t 2
0°C
(h
ou
rs)
Total germination after controlled deterioration (%).
MGT and seed age: oilseed rape
High MGT
Slower germination
Long lag period
Low MGT
Faster germination
Short lag period
More aged
Less aged
As seeds age, the lag period to RE increases (higher MGT)
2012: Seed Sci. &Technol., 40, 413-424
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MGT and seed age
Seed age measured by AA or CD negatively correlates ( r) and predicts (R2) MGT
significantly (mostly p< 0.01 or p< 0.001)
Species (number of lots) r R2
Oilseed rape (9) -0.92 0.85
Maize (9) -0.79 0.62
Radish (10) -0.89 0.80
Cotton (17) -0.91 0.83
Pepper (11) -0.97 0.95
Watermelon (10) -0.95 0.90
Melon (10) -0.75 0.57
Cucumber (9) -0.94 0.88
Viola (9) -0.91 0.83
The more aged the seed, the longer the lag period
For references see STI (2012), 142, 31-35
Relates to ageing tests
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0
20
40
60
80
100
0 24 48 72 96 120 144
Time from set to germinate (h)
Germ
ination (
%)
F
H
I
Lag period
Longer lag period
Germination progress curves
MGT = Ʃnt / Ʃn
n = number of newly germinated seeds at time t
t = days from when seeds were set to germinate.
MGT= Mean length of lag period
• Mean length of lag period differs between commercial seed lots
• Ageing increases MGT and spread of timing of germination
Ageing
Maize
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MGT and seed performance (vigour)
• Rate of emergence
• Final emergence
• Spread of emergence
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MGT predicts rate of and final emergence: oilseed rape
y = -5.88x + 271.79
R² = 0.930***
y = -2.34x + 165.39
R² = 0.745**
20
40
60
80
100
30 35 40 45
Fie
ld e
mer
gen
ce (
%)
MGT at 20°C (hours)
Final emergence
Low MGT
Short lag period
Early emergence
High final emergence
High MGT
Long lag period
Late emergence
Low final emergence
7 day emergence
2012: Seed Sci. & Technol., 40, 413-424
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MGT (R2) predicts rate of, and final, emergence in many species.
All R2 significant: mostly at P< 0.01 or P<0.001
Longer delay to RE; higher MGT
Slower and lower emergence
Low
vigour
Species (lots) Rate of
emergence
Final emergence
(%)
Oil seed rape (9) 0.93 0.75
Radish (9) 0.92 0.86
Maize (9) 0.62 0.72
Cotton (13) - 0.83
Pepper (11) 0.94 0.45 (modules)
Watermelon (10) 0.83 0.81
Melon (10) 0.74 0.70
Cucumber (9) 0.76 0.64
Viola (9) 0.91 0.92
References, see STI (2011), 142, 31-35
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Maize: MJGT and MGT relate to seedling size and uniformity in lab
(14 d, 13oC)
MJGT = 3.52 days
MGT = 4.80 days
MJGT = 4.82 days
MGT = 5.84 days
Timing and spread of emergence: maize
High MGT indicates slower and more spread emergence
Relates to seedling growth tests
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Timing and spread of emergence
Important for vegetable production from modules
Cabbage High MGT Low MGT
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SO FAR:
MGT from germination curves relates to vigour
BUT:
Frequent RE counts not convenient for a routine test
Are there alternatives?
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M.-H. Wagner – Seed Symposium – IPK Gatersleben 11/04/18
Automated monitoring since 2005
• Digital imaging upon Jacobsen incubators
• High throughput, thermal precision, repeatability
• Top of paper germination
Demilly et al., 2014
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30 hours
Differences seen in
early radicle emergence
counts (30 hours)
Oilseed rape
Automated germination progress curves (GEVES)
2012: Seed Sci. & Technol., 40, 413-424
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y = -0.17x + 49.92
R² = 0.910***
30
35
40
45
40 50 60 70 80 90 100
MG
T a
t 2
0°C
(h
ou
rs)
Radicle emergence (%) at 30 hours
Oilseed rape: Single count of RE predicts MGT
2012: Seed Sci. & Technol., 40, 413-424
Low MGT,
Short lag period
High RE
High MGT,
Long lag period
Low RE
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Early single counts of RE (top of paper) predict MGT in 17 species
Automated digital imaging (GEVES)
All significant;
13/17, R2 >0.90
Temp Species No. of
lots
analysed
MGT (h) range RE count (h)
best related to
MGT
Highest
R2
20o C
OSR 68 28-60 34 0.87
Maize 35 49-78 60 0.83
Radish 6 29-56 40 0.98
Wheat 15 29-72 50 0.97
Cabbage 40 23-60 36 0.90
Onion 32 33-103 72 0.87
Tomato 56 52-125 82 0.92
Sugarbeet 17 49-75 64 0.67
Lucerne 30 18-39 30 0.91
Lettuce 8 23-28 24 0.94
Carrot 8 41-69 56 0.97
25oC
Sunflower 7 28-38 36 0.97
Eggplant 8 76-96 90 0.99
Turnip 8 16-20 18 0.97
Chicory 8 23-31 40 0.93
Parsley 8 123-161 130 0.95
Cotton 5 46-61 52 0.99
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y = 1.07x - 24.75
R² = 0.961***
y = 0.40x + 48.67
R² = 0.713**
20
40
60
80
100
20 40 60 80 100
% R. E. 30h at 20°C
Fie
ld e
mer
gen
ce (
%)
7d
Max
Oilseed rape: Single count of RE predicts field emergence
2012: Seed Sci. & Technol., 40, 413-424
Low RE
Slow emergence
Lower final emergence
High RE
Faster emergence
Higher final emergence
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Species Lots (n) Time of RE count r R2 Significance
( p< )
Maize 9 66h , 20oC
6d, 13oC
0.76
0.85
0.58
0.72
0.02
0.01
Oilseed rape 9 30h, 20oC 0.84 0.71 0.01
Radish 11 48h, 20oC 0.95 0.90 0.001
Cotton 10 3d, 18oC 0.87 0.76 0.001
Rice 13 54h, 25oC 0.90 0.81 0.001
Watermelon 11 68h, 25oC 0.81 0.66 0.01
Melon 10 44h, 25oC 0.74 0.55 0.05
Cucumber 10 48h, 25oC 0.84 0.70 0.05
Viola 9 48h, 20oC 0.82 0.67 0.01
Also seen in wheat, alfalfa, onion and forage crops
Early single RE counts
correlate ( r) and predict (R2) final emergence in many species
For rice: Luo et al. (2017), Seed Sci. & Technol., 45, 1-8
All other species see STI (2011), 142, 4- 48
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RE vigour test and ISTA: Validation then ISTA Rules
• Test relates to performance (vigour)
• Convenient for routine use – single early RE count
• Repeatable
• Reproducible
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y = 0.86x + 16.95
R² = 0.95***
0
20
40
60
80
100
0 20 40 60 80 100
Lab
ora
tory
B
rad
icle
emer
gen
ce (
%)
Laboratory A radicle emergence (%)
y = 1.012x - 2.65
R² = 0.96***
0
20
40
60
80
100
0 20 40 60 80 100
Lab
ora
tory
C r
adic
le
emer
gen
ce (
%)
Laboratory A radicle emergence (%)
y = 1.126x - 19.82
R² = 0.94***
0
20
40
60
80
100
0 20 40 60 80 100
Lab
ora
tory
C r
adic
le
emer
gen
ce
( %
)
Laboratory B radicle emergence (%)
Repeatability of RE in different laboratories : oilseed rape
Validated for oilseed rape:2014
ISTA Proficiency test 2016
57 labs; 70% satisfactory
Matthews, Wagner, Kerr, McLaren and Powell (2012) Seed Sci. & Technol, 40, 413-424
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Species Germination
medium
Replication Germination
temperature
Criterion of radicle
emergence
Timing of radicle
emergence count
Zea mays Paper towels 8 replicates
of 25 seeds
20 ± 1oC
or 13 ± 1oC
Production of 2mm
radicle
66h ± 15 min at 20 ± 1oC
144h ± 1 h at 13 ± 1oC
Brassica
napus
Pleated
papers
2 replicates
of 100 seeds
20 ± 1oC Appearance of a
radicle after breaking
through the seed
coat. Seeds in which
the seed coat has
split, but no radicle
has emerged, must
not be included.
30h ± 15 min
Raphanus
sativus
Top of paper 4 replicates
of 50 seeds
20 ± 1oC Production of 2mm
radicle
48h ±15 min
Specific directions for RE test in ISTA Rules
Note: alternative germination
media not acceptable for RE test Precision essential
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Lag period and the cold test: maize
SLOW FAST
y = 0.86x + 11.62
R² = 0.96
p < 0.001
0
20
40
60
80
100
20 40 60 80 100
Germination after 6 days at 13°C
Ge
rmin
ati
on
in
co
ld t
es
t
(% n
orm
al s
ee
dlin
gs
)
Low RE
Long lag period
Low cold test germination
High RE
Short lag period
High cold test germination
RE
Seed Sci. & Technol.(2010), 38, 379-389
RE relates to cold test
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Significance of the lag period
T.A Villiers, 1970’s : Lettuce
Dormant seeds stored in the dark for 12 months
Dry seeds Fully imbibed seeds (in air, moist filter paper)
Germinated in light Germinated in light
Low germination (RE)
Many chromosome aberrations
Many abnormal seedlings
High germination (RE)
Few chromosome aberrations
Few abnormal seedlings
Villiers hypothesis: metabolic repair of membranes and DNA when stored imbibed
D. Osborne: 1980’s and 90’s: rye
• Evidence of DNA repair in lag period before radicle emergence
• Supporting and detailed evidence for DNA repair from Arabidopsis mutants
(Waterworth et al., 2007 to date)
• Some evidence for membrane repair
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y = 0.492x + 11.83
R² = 0.850***
30
40
50
60
70
80
90
100
40 50 60 70 80 90 100 110 120 130 140 150
MG
T (
hours
)
EC µS cm-1 g-1 after 3h (EC3)
A
Evidence for membrane damage
Radish and bulk EC 100 seeds in 40ml water
Longer lag period (high MGT)
In lots with higher leakage (EC)
Mavi et al., (2016) Seed Sci. & Technol., 44. 393-409
RE relates to EC test
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Evidence of membrane damage and repair
Radish: single seed EC and RE performance
(Amalgamated data from 20 seeds from each of 10 seed lots)
EC measured after 5 hours in water at 20oC, followed by germination on moist paper at 10oC
All
seeds
Dead
seeds
Seeds that produce: Seed producing normal
seedlings with:
Abnormal
seedlings
Normal
seedlings
Early RE
<66h
Late RE
>66h
Number of seeds
199 22 14 165 73 92
Mean EC (5h)
6.88 14.24 11.99
(MGT: 122h)
5.79
(MGT: 91h) 4.25 6.26 * **
Also:
• 5h hydration in air drying back
• Significant reduction in leakage and lag period
• Evidence of metabolic repair.
Mavi et al. (2016) Seed Sci. & Technol., 44, 393-409
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Concluding comments
• As seeds age the lag period to RE increases (higher MGT)
• As MGT increases – Rate and final emergence decrease
– Longevity in store decreases
• Higher MGT (longer lag period) smaller and less uniform seedlings
• An early count of RE (manual or automated) – Predicts MGT
– Predicts seed vigour – validated for maize, oilseed rape, radish
– Evidence for its potential for many other species
• Hypothesis: as seeds age, the need for repair and lag period increase
• Hypothesis explains connections with other vigour tests – AA, CD, seedling growth, cold and EC tests